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Type of publication: Master Thesis
Type of document:

Year: 2012

Authors: Stadlmann, Valerie

Title: Establishment of a novel system for the quantitative determination of diamine oxidase.

Other title: Entwicklung eines neuen Systems für die quantitative Bestimmung von Diaminoxidase

Source: Master Thesis, Vet. Med. Univ. Wien, pp. 70.


Advisor(s):

Razzazi-Fazeli Ebrahim

Reviewer(s):
Staniek Katrin

Vetmed Research Units:
VetCore


Graduation date: 26.09.12


Abstract:
Aim of this master thesis was to develop a novel assay for the quantitative determination of diamine oxidase (DAO) in biological fluids. DAO is a coppercontaining, secretory enzyme which plays a major role in the metabolism of food-borne biogenic amines (e.g. histamine). A misbalance of accumulated amine concentrations and DAO quantity or activity may lead to adverse food reactions like histamine intolerance (HIT). Current diagnostic methods for HIT are based on the measurement of human serum DAO activity, which shows a rather poor correlation to the clinical presentation of HIT. A quantitative test which measures the actual amount of DAO is therefore desired and would improve the diagnosis of HIT. In this study human placental diamine oxidase was isolated and purified via ammonium sulfate precipitation, Hydrophobic Interaction Chromatography (HIC) and Cellufine Sulfate (CS) Affinity Chromatography. Purification quality was characterized with BCA Total Protein Determination, Radioextraction assay (DAO-REA®) and SDS-PAGE. Purified diamine oxidase was used as immunogen for the immunization of rabbits. The IgG fraction of rabbit anti-DAO antisera was purified. Two ELISA systems were tested: In a competitive ELISA assay, CS-purified DAO served as sample and was competing with HIC-purified DAO for rabbit anti-DAO IgG. Under optimized conditions, a buffer containing guanidine-hydrochloride and HEPES was used and incubation conditions with the primary rabbit antibody were adjusted to 37°C and 3 h. CS-purified DAO dilution range was assessed between 0-14 nM, a competition with 100 ng HIC-purified DAO/ well was detectable between 3-14 nM of CS-DAO. The second assay was a Sandwich ELISA: rabbit anti-DAO IgG was coated on ELISA plates 70 ng/ well. HIC-purified DAO served as sample and was detected with biotinylated rabbit anti-DAO IgG. In optimized sandwich ELISA, HIC-purified human DAO was detectable in a dilution range of 0.6-5 μg/ml total protein using biotinylated rabbit anti-DAO IgG in a 1:3,000 dilution in a buffer containing guanidine-hydrochloride and HEPES. Streptavidin conjugated to horseradish peroxidase was diluted 1:30,000 in commercially available conjugate stabilizer. 1% Polyethylene glycol 6000 proved promising as blocking agent for this ELISA. The here developed ELISAs await validation and it has to be noted that both ELISA systems were characterized using pre-purified human placental DAO instead of native physiological samples.


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